In the adult, hematopoiesis is maintained by the constant regulated production of billions of mature blood cells derived from a small population of relatively quiescent hematopoietic stem cells (HSC) located in the bone marrow. These cells have the potential to undergo self-renewal to maintain their numbers and to produce cells of all the hematopoietic lineages. Proliferation and differentiation of HSC produces a heterogeneous compartment of actively dividing cells called hematopoietic progenitors. With progressive cell divisions, these progenitors have limited self-renewal capacity and become committed to the various blood cell lineages i.e. white blood cells, red blood cells and platelets.
Since their introduction more than 30 years ago, colony assays have been used extensively for research and clinical applications of hematopoietic cells in particular. These include identification of stimulatory and inhibitory growth factors, supportive diagnostic assays of myeloproliferative disorders and leukemias, and evaluation of the hematopoietic proliferative potential of bone marrow (BM), cord blood (CB) and mobilised peripheral blood (MPB) samples for clinical transplantation. Hematopoietic progenitor cells will proliferate and differentiate in vitro to produce distinct colonies in semi-solid media. Culture conditions that promote such colonies have been optimised and standardised offering a reproducible functional assay for hematopoietic progenitors. This assay has been called the colony forming cell (CFC) assay. The CFC assay has become the benchmark functional assay to assess the ability of various hematopoietic cell sources to divide and differentiate, especially following ex vivo manipulations including T-cell depletion, CD34.sup.+ cell enrichment, gene therapy protocols and cryopreservation. The CFC assay is also used to screen novel drug candidates for potential toxicity on hematopoietic cells.
Hematopoietic CFC assays are used to quantify multi-potential progenitors and single lineage restricted progenitors of the erythroid, granulocytic, monocyte-macrophage and megakaryocytic pathways. The most commonly used procedure involves the plating of a single cell suspension into semi-solid nutrient medium supplemented with the appropriate combinations of cytokines that supports the proliferation and differentiation of individual progenitor cells into discrete colonies containing recognizable progeny. The CFCs are classified and enumerated based on morphologic recognition of mature cells within the colony in situ by light microscopy. The number of colonies obtained should be linearly proportional to CFC content of the input cell suspension provided that a sufficiently low number of cells are plated. In general, under ‘optimal’ assay conditions, colonies containing cells of two or more lineages (mixed colonies) arise from a more primitive progenitor than those containing cells of a single lineage. In addition, more immature progenitors generate larger colonies and require a longer period of time in culture to allow maturation of the cells within the colony.
Colony identification on the basis of size and morphology is inherently subjective and there can be considerable variation in the determination of absolute CFC (Bacigalupo et al. Bone Marrow Transplantation (1995); Torres et al. Blut (1985)). Inconsistent scoring can result from differences in the criteria for colony identification between groups and inexperience of laboratory personnel (Burger et al., Transfusion (1999); Lumley et al. Bone Marrow Transplantation (1999); Lamana et al., Bone Marrow Transplantation (1999)). When training new personnel, comparative counting with experienced staff provides the most useful form of instruction and should be continued until an adequate level of proficiency is attained. Even for accomplished staff, cross-comparisons once a month of counts with common set of progenitor cultures is recommended to maintain reproducible scoring. In addition, different types of colonies can only be counted at the end of a lengthy culture period, typically 14-16 days, when colonies have sufficiently matured.